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among tissues was evaluated by the measure-
ments of coef
all analytes by a similar degree, the result of the
CITP stacking process is that major components
may be diluted, but trace compounds are concen-
trated.
55
Furthermore, CITP offers the bene
cient of variation (CV) and the
Pearson correlation coef
cient.
50
Analytical
reproducibility of relative protein abundance
was determined to exhibit a Pearson R
2
value
greater than 0.99 andaCVof 14.1%. TheCIEF pro-
teomic platform was capable of measuring
changes in protein expression as low as 1.5-fold
with con
ts
of speed and straightforward manipulation/
switching between the stacking and separation
modes in transient CITP/CZE. Transient CITP/
CZE further provides seamless combination
with nano-RPLC (
Figure 2
) as two highly
resolving and completely orthogonal separation
techniques critically needed for analyzing human
saliva
dence as determined by t-test.
The protein expression pro
les from two
distinct ovarian endometrioid tumor-derived
cell lines have been compared using CIEF-based
multidimensional separations coupled with
ESI-MS/MS.
51
Differentially expressed proteins
were further investigated by ingenuity pathway
analysis to reveal their association with impor-
tant biological functions and signaling pathways
such as the P13K/AKT pathway. The results
illustrated the utility of high-throughput proteo-
mic pro
and mouse
brain mitochondrial
proteomes.
56,57
The ultrahigh resolving power of transient
CITP/CZE as the
first separation dimension
has been demonstrated by signi
cantly low
peptide fraction overlapping for the analysis
of protein expression within glioblastoma
multiforme-derived cancer stem cells.
58
Approx-
imately 89% of distinct peptides were identi
ling combined with bioinformatics tools
to provide insights into the mechanisms of dereg-
ulation in neoplastic cells.
In addition to CIEF, microscale in-solution
IEF was employed as the
ed
in only a single CITP fraction. In contrast,
a high degree of peptide overlapping in strong
cation exchange (SCX) chromatography, as the
first separation dimen-
sion for the fractionation of intact proteins
according to their isoelectric points, followed
by proteolytic digestion with trypsin and subse-
quent CZE peptide separation coupled off-line to
MALDI-TOF/TOF-MS.
52
The platformwas used
for the analysis of human follicular
first separation dimension of the multidimen-
sional protein identi
cation technique (MuD-
PIT),
59
was observed with at
least 40% of
carryover peptides
ed in
previous salt gradients. A high degree of peptide
overlap in SCX unnecessarily burdens the subse-
quent nano-RPLC separation and greatly
reduces the overall peak capacity in a multidi-
mensional separation system.
The CITP proteomic platform provided signif-
icant enhancements in total peptide, distinct
peptide, and distinct protein identi
that were identi
fluid with
clinical implication. A total of 73 unique proteins
were identi
ed, including mostly acute phase
proteins and proteins that are known to be exten-
sively involved in follicular development.
cations
over a corresponding MuDPIT run by 119%,
192%, and 79%, respectively.
58
The CITP proteo-
mic technology, equipped with selective analyte
enrichment and ultrahigh resolving power,
further accomplished superior coverage in key
pathways than that of the MuDPIT. For example,
many biologically relevant proteins, including
MKP, the Raf family, and Src in the ERK/
MAPK pathway, were identi
Transient Capillary Isotachophoresis/
Capillary Zone Electrophoresis
Besides proteome complexity, the greatest bio-
analytical challenge facing comprehensive pro-
teomic analysis, particularly in the identi
cation
of low-abundance proteins, is related to the large
variation of protein relative abundances. For
example, the protein concentration dynamics
range from 10
6
-fold in cells to 10
12
-fold in
blood.
53,54
ed only by the
In contrast to universally enriching
CITP technology (
Figure 3
).
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